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1 A.M. ZOOLOGIST, 2: (1962). CELLS OF THE BLOOD AND COELOMIC FLUIDS OF TUNIGATES AND ECHINODERMS WARREN ANDREW Department of Anatomy Indiana University School of Medicine INTRODUCTION The cells of the blood and coelomic fluid in various groups of invertebrate animals present a surprising degree of variety and complexity. Since these cells as a whole seem to correspond to the cells of the blood, the free cells of lymphoid tissue, and the wandering cells in the tissues of vertebrates, a clearer understanding of their structure and activities is not only of much intrinsic interest but may also be expected to throw much light on the unsolved problems pertaining to such cells in vertebrates. The present paper describes some of the features of these cells in two important groups of animals, the echinoderms and the tunicates, the latter showing many features which lead us to believe them not far removed from the ancestors of the vertebrates. The observations reported here have been made primarily on living cells with the aid of bright contrast phase microscopy. The cells have been studied in cover-slip preparations or within minute fragments of tissue from various parts of the body, including gills, intestine, gonads and body wall. In such preparations, clotting is a very slow process and in some instances, as in the blood of tunicates, may not even occur. Notes were kept of observations on groups of cells and individual cells over periods of as much as 24 hours; drawings were made of many; and photomicrographs obtained of some. These studies were carried out at the Bermuda Biological Station for Research during the summers of 1959 and The author is sincerely grateful to Doctor William Sutcliffe, Director of the Station, and his staff, for many courtesies. TUNICATES General Features of Blood Cells and Plasma. This section is restricted to the hemocytes of adult ascidians. Thaliaceans, appendicularians (said not to have blood cells), and larval ascidians are not considered. Several types of blood cells are present in larval ascidians (Andrew, 1961). In the adult ascidians, blood cells are so numerous that centrifugation yields a rich sediment of cells; these were very early subjected to macrochemical analysis (Henze, 1911, 1912, and 1913). Henze showed the presence of a chromogen in the blood of several species and demonstrated that this chromogen contains a high content of vanadium. He associated this vanadium-containing chromogen with his so-called "mulberry" cells (maulbeerformigen Zellen), and he verified the above association in Phallusia, Ascidia rnentulata, A. fumigata, Ciona intestinalis, and Diazona violacea. In Cynthia papillosa, in which he could not demonstrate vanadium, there were no mulberry cells. Henze characterized the mulberry cells chemically as reacting with osmic acid to become deep black, and undergoing other specific reactions. He also found the structure of these cells, that is, the individuality of the "berries" within them, to become clearer under certain conditions, as in the presence of a trace of ammonia. These cells are strongly acid, as shown by an indicator such as methyl red. Henze distinguished the "mulberry" type of cell from another, somewhat larger round cell. These cells probably represent, (285)

2 286 WARREN ANDREW at least in part, the "macrophages" of later authors, as seen in rounded forms under conditions not favorable for ameboid activity. It appears that there is no loosely bound oxygen present in ascidian blood, and that the chromogen is not an oxygen carrier. There is evidence that the vanadium chromogen functions as a catalyst, particularly as an activator of oxygen. The free sulfuric acid may act to prevent an auto-oxidation of the chromogen. The concentration of the acid in the blood cells, although they are suspended in a neutral plasma, is amazingly high about three per cent! This is about twenty times as much SO 3 as is present in the sea water. Since the mulberry cells alone appear to be acid in content, and since they constitute about 60% of the total number of cells, the actual acid content in these specific cells is probably between five and six per cent. In Ascidia mentula the tunic also gives a strong acid reaction, but in Phallusia, although the same cellular content seems to occur, namely large vesicular "bladdercells," the tunic shows no trace of free acid. The plasma, or fluid portion of the blood, in which the cells are suspended, is very similar to sea water, but differs in the proportions of some of the chemical components. The plasma has only about onehalf as much SO 3 as does sea water, and the conclusion seems natural that SO 3 is being taken up by the cells to give them their high content of free sulfuric acid. The plasma is low in protein content, and this has led various authors to surmise that some of the blood cells are carrying nutrients to the other tissues. "Clotting" in the tunicates consists in the agglutination of cells. In Ascidia nigra, when the blood, which is of a conspicuous green color, flows out into sea water, green strands soon form and maintain themselves for some time. There appears to be no clotting mechanism involving the plasma. Distinct types of cells. Cuenot (1891) pointed out that the blood cells in tunicates seem to be unequally distributed in the circulation. For example, the orange cells of Ascidia mentula are scarce in the fine vessels and tissue fluid, but seem to be concentrated in the heart and great vessels. In some ascidians, e.g., Molgula, Cuenot thought that cells corresponding to vertebrate red cells, are present. These are irregularly spheroidal cells up to 45 /x in diameter, often with eccentrically located nuclei. These cells are elastic, so that in spite of their large size they can go through lumina of small vessels. Later authors appear not to have worked with the blood of those forms which contain such cells. Seeliger and Hartmeyer (1911) described heaping up of blood cells in the blood vessels, particularly in the smallest branches. They also found many evidences both of degeneration of blood cells and of division leading to their multiplication. Kollman (1908) studied blood of a number of species, and on the basis of the great variability of the blood cells in size, form, mass, inner structure, and other features, felt that Cuenot's classification was somewhat artificial. The blood cells of individual species of tunicates and of groups of species were described in papers by Fulton (1920) and George (1926, 1930, and 1939). More recently, Sabadin (1953) and Endean (1955) have contributed important papers. Our own previous study (Andrew, 1961) was based on the blood of some of the same species studied by Fulton and by George, but we hope that in the future it will be possible to extend our observations to Pyura, studied by Endean, and Bolryllus, studied by Sabadin. We shall give here a general description of the blood cells, in the light of past work and of our own studies, referring the interested reader to the original papers for more detailed discussion, particularly of controversial points. A division of the cell types into two main categories of colored and colorless ones seems at first sight to be a natural one. In such a division, the colored group includes:

3 CELLS OF FLUIDS IN TUNICATES AND ECHINODERMS FIG. 1. Ascidia nigra, phase contrast. Blood showing many green cells, several vacuolale cells, and one blue cell (in the center of the field). Note the cvtoplasmic center in the green cells which is devoid of the included green substance. X ca FfG. 2. Ascidia nigra, phase contrast. Blood showing four somewhat irregularly shaped macrophages, a few vacuolate cells and a number of green cells. In this specimen the included material in the green cells shows more o a spherular condition, probably because of longer standing. X ca green cells are motile. We can affirm from our own observations on Ascidia nigra, Clavelina picta and Ecleimiscidia turbinnla that these cells progress by ameboid motion. While the motion may be fairly slow in cover-glass preparations, it is rapid indeed in the tunic of some species, as in Clavelina picla. The greenish substance appears as a viscid mass flowing within the cytoplasm, and moving cells show very sharply the distinction between the living protoplasm and the "included" material. The other two types of colored cells in the blood of Ascidia nigra are present only blue, orange, green, and brown or reddishbrown types. The colorless ones include small ameboid leucocytes (lymphocytes), large ameboid leucocytes (macrophages and reticulated leucocytes), and quiescent colorless cells which are of a vacuolar nature, generally with one large vacuole, but sometimes with two, three or more such vacuoles. While this division into colored and colorless cells may seem a natural one, it can be misleading in some respects. For example, there is a type of cell which resembles the green cell in all ways except one it is not green! These cells have been classed by George (1939) as "colorless cells of green cell type." Another difficulty is the fact that there is at least a good probability that the colored cells are derived from colorless ones, although some authors believe that they represent cell types of completely different origin from the leucocytes. In Ascidia nigra, the green cells are the most numerous of all of the cell types, and generally constitute one-half of the total number of cells. They give the blood a bright green color. With the oil immersion lens, each cell of this type is seen to have in it a number of green bodies which are pressed closely together, generally leaving a small central clear area and a very nai"row periphery of cytoplasm. The green bodies, while appearing as wedges, seem to become spheroidal readily, perhaps because of release of pressure. A certain proportion of the cells will show green spherules in freshly drawn blood (Figs. 1 and 2). A slight pressure applied to the cover-glass will bring out sharply the individual nature of the intracellular bodies (Figs. 3 and 4). This makes the green cells appear very similar to the spherule cells of the echinoderms which we have described and pictured (Fig. 10). The green cells are undoubtedly also the "mulberryshaped cells" of Henze which contain the vanadium chromogen. There was some difference of opinion among earlier authors as to whether the 287

4 WARREN in small numbers, from one to three per cent of the "differential" count. They are the orange cells and the blue cells. The orange cells resemble the green cells more than they do the blue ones. In fact, it seems probable that they are of the same general type as the green cells and therefore similar to spherule cells of the echinoderms. The orange substance is included material and often seems to occur as separate "bodies." They rarely become spherular. The "bulging" contents give an irregular appearance to the orange cells. As with the green cells, there has been difference of opinion as to the existence and type of motility of these cells. Fulton (1920), for example, said that they are active swimmers. Our own observations give no evidence of rapid swimming motion, but we have seen the formation of blunt pseudopods from them, perhaps indicating that under proper conditions, as in the tissues, they may progress by ameboid motion. The third type of colored cell, the blue cell, is present only in a small number of species of tunicates. In Ascidia nigra, the form best known, the blue cells of the blood are very similar in their general morphology and in their motion to the large ameboid leucocytes or "macrophages" of this animal. The only way in which they seem to differ from the latter cells is by the presence in the cytoplasm of the blue granules and by the absence of phagocytic activity. The included blue material of the blue cells is in the form of bodies which present a considerable degree of variety in size and form, ranging from coarse, somewhat angular granules or elongated bars to very tiny bodies. In moving cells this material does not show the viscid property exhibited by that in the green cells. The ameboid leucocytes, or motile colorless cells, include smaller, often rounded cells which in their nucleocytoplasmic ratio and general structure are similar to the lymphocytes of vertebrates, and which may well be designated as lymphocytes, and the larger, actively ameboid leucocytes which we may speak of as macrophages. The latter show a range of appearances which seems to depend largely on their degree of phagocytic activity. The macrophages which are not actively phagocytic have a relatively inconspicuous ectoplasm. They move along in a distinctive fashion, almost always having a long, thin, pointed pseudopod stretching out in front of the cell. This special pseudopod moves slowly to and fro, as though "feeling its way." It seems to have little to do with the actual forward motion, which is accomplished by more conventional blunt pseu- FIG. 3. Ascidia nigra, phase contrast. A field in which many green cells and a few vacuolate cells are present. Note the somewhat wedge-shaped inclusion bodies of the green cells and the sharp outline of the cell body. X ca FIG. 4. Ascidia nigra, phase contrast. Specimen in which slight pressure was put on the cover-slip. The inclusion bodies stand out more sharply and the cell outlines are less marked. X ca ANDREW

5 CELLS OF FLUIDS IN TU.NICATES AND ECHINODERMS \ FIG. 5. Single trephocyte in body wall. Note the relatively small, dark nucleus. In the living condition, all trephocytes in the body wall are actively motile. Masson's irichrome stain. X ca FIG. 6. Trephocyte in section of body wall stained by Gomori's Aldehyde. The cell appears as a large dark mass, densely stained throughout. X ca dopodia. This type of motion is the same as lhat of the blue cells (Fig. 5). Arnold (1959) has found a temporary structure very similar to our "pointed pseudopod," in the hemocytes of a cockroach, lilabenis giganteus. Of such pseudopodia in the roach, he says (p. 374): "They were turgid, tactile, and especially when extended in a clear space, oscillated slowly within an arc of approximately 15 degrees"; and again: "They were the means by which the cell's movement was directed, particularly into narrow spaces where the typical, less turgid and apparently less sen- sitive pseudopodium would scarcely penetrate." It appears, then, that the blue cells and macrophages of tunicates often exhibit a type of pseudopodium which may be described as a temporary sensory organ of the cell! In many macrophages, particularly in certain specimens and apparently more often in those individuals which have been kept longer in the tanks, there is an altered appearance. In such cells the ectoplasmic portion is sharply distinguished from the endoplasm and appears as clear, wide sheets surrounding it. The endoplasm contains granules and lipid droplets of varying size. Such cells are very active but less frequently show the pointed pseudopod described above. The other type of colorless ameboid cell may be called the colorless compartment cell (George, 1939) or the reticulated ameboid cell. It is marked by sharp "septalike" lines in the cytoplasm. This type of cell moves slowly, and its ectoplasmic border is narrow and inconspicuous. It does not appear to contain phagocytized material. The dark bands form a striking network in the generally clear, light gray cytoplasm. The other colorless cells of the blood are those which have been described by us (Andrew, 1961) as the vacuolar cells, and which we subdivided into unilocular, bilocular, and trilocular vacuolar. This terminology indicates that they contain an "included" material, which is present within one, two or three vacuoles or bodies. In the unilocular vacuolar cells a small concretion is usually present in the vacuole (Fig. 10) and its constant migration, probably a type of Brownian movement, would indicate that the contents of this single vacuole are in a fluid state. In bilocular and trilocular cells we often see the individual vacuoles appearing somewhat like the blastomeres of an embryo or indeed, like the bodies present in a green cell, the three bodies, in particular, appearing "wedged in" together. The unilocular vacuolar cell Photomicrographs of staining sections of body wall of Stichopus badionotus. 289

6 290 WARREN ANDREW lets, these structures are cell fragments which continue to live, and also like the platelets they are characterized by a markedly adhesive nature. Fixed tissue counterparts of the blood cells of tunicates. The blood cells and coelomic corpuscles of many invertebrates are to be found wandering widely in the tissues of the body. In the tunicates there are many blood cells moving amid the various tissues, but here an added feature is conspicuous the presence of fixed tissue counterparts of the blood cells. These are non-motile cells which correspond to, and almost certainly are derived from, the blood cells. Prominent among the fixed tissue counl'hotomiciographs of trephocytes from intestine and terparts are the blue cells of the test, as in body wall of Slichopus badionolus. Ascidia nigra. These cells are stable elefig. 7. Trephocyte from the intestinal submucosa. ments, often showing extended processes, Note again the small nucleus. A number of inclusion bodies can be seen in this cell bulging the cyto- distributed through the thick tunic. Along plasm in places. Nfasson's trichrome stain. X ca. with these cells are colorless or grayish 816. cells, which resemble vertebrate fibroblasts FIG. 8. Another cell from intestinal submucosa, and which might be thought of as countershowing a smaller nucleus, and apparently less included material. Masson's trichrome stain. X ca. parts of the larger ameboid cells or macro816. phages. FIG. 9. Trephocyte from body wall. Masson's trithe orange cells of the blood are reprechrome stain. X ca sented by conspicuous fixed tissue counteroften shows a distinct cytoplasmic "cap" or parts in some species of tunicates, as in Eccrescent of cytoplasm, as described by us teinascidia turbinata, where the tunic shows numerous very large orange cells and earlier by Fulton (1920). A peculiar feature of the unilocular cells such as the "pigment cells" of this species. is the presence of actively undulatory These cells contain distinctive fusiform orbeaded appendages. These vary in length ange bodies. from about equal to the diameter of In the same way, the purplish-red blood the cell (7 to 9 ju) up to 4 or 5 times this cells of Clavelina picla have their counterand in exceptional cases up to 80 or 90 p. parts in the pigment cells which form the As a part of the cell, the beaded append- beautiful ring on the oral siphon and the age will continue to undulate for many streak along the endostyle of this species. hours in cover-glass preparations and even Fixed tissue counterparts of the colorless when detached, fragments composed of sev- compartment cells of the blood have been eral beads may continue motion for over an identified in the tadpoles of Ecteinascidia hour. The beads are markedly adhesive turbinata as fibroblast-like cells with sharpand broken portions of the appendages will ly reticulated cytoplasm (Andrew, 1961). fasten to the underlying glass. While the homology is not clearly estabwhile it is premature to assign a func- lished, there is a similarity between the tion to these structures, there is a super- three kinds of vacuolar cells in the blood ficial resemblance to the blood platelets in Ascidia nigra (and in other species) and seen in many vertebrates. Like the plate- the large vacuolar cells of the test in this

7 CELLS OF FLUIDS IN TUNICATF.S AND ECHINODF.RMS 291 functions of locomotion and respiration. Cells of various kinds, particularly wandering cells, can be found in any one of these fluids and probably pass freely from one to the other. This does not preclude entirely the importance of a distinction between the fluids, as there are some definite barriers set up between them which apparently involve substances and even cells, at least of the non-motile kind, as we shall see later. The coelomic fluid is abundant in the sea-urchins, and its chemical characteristics have been described. Jt gives a neutral reaction, and contains a number of mineral salts and small quantities of organic matter, including fats and protein. The blood of echinoderms seems to contain protein in a small quantity, similar to that of the coelomic fluid, while there is still less in the water vascular system. Botazzi (1925), however, believed that the quantities of organic material are extremely small if the fluid is well freed of cells. Thus, in a holothu11 roidean, Holothuria lubulosa, the protein FIG. 10. Blood of Ascidia nigra, phase contrast. Near the center of the field is a unilocular vacuo- was g per 100 ml, and in an echinoid, Sphaerechinus granular is, it was g late cell showing the concretion which is present in most, if not all, of these cells. The concretion is per 100 ml. very darkly outlined here. A group of green cells is Botazzi believes that there exists during seen in the lower part of the figure. X ca FIG. 11. Blood of Mellita quinquiesperforata,\>\\asc the progressive development of the blood contrast. A large colorless spherule cell is seen. (by "blood" meaning to say all the inner These cells are very active in cover-slip preparabody fluids of an organism) in the animal tions. X ca kingdom a series in which such fluid first black ascidian. These test cells, while gen- represents primarily a solution of inorganic erally unilocular, are at times bilocular or salts. In its qualitative and quantitative trilocular and, like the vacuolar cells of chemical composition and in its physicothe blood, a small mineral concretion is a chemical properties such a solution is very similar to sea-water, and, while it carries common feature of the cell. in suspension the various cells, does not ECHINODERMS carry colloidal material to any extent. In General features of the body fluids. (he higher invertebrates and in the vertethere are three types of body fluids pres- brates the blood is clearly a colloidal susent in the echinoderms: (1) the coelomic pension. In the molluscs and arthropods fluid, contained in the body cavity, (2) the the blood begins to become a colloidal soblood, contained in a system of blood ves- lution, but at the same time retains, at sels, and (3) the fluid of the water vascular least in the marine forms, many of the system, the apparatus which includes the properties of sea-water. Yet even in the madrepore, a system of canals and the tube molluscs and crustaceans the protein is of feet, and which appears often to combine the nature of a respiratory colloid and

8 292 WARREN ANDREW thus, while outside of cells, is analogous to the hemoglobin in the erythrocytes of vertebrates. Morphology and behavior of the cells in echinoderms. In echinoderms, particularly among the Holothuroidea, respiratory pigment or protein occurs inside blood cells. This evolutionary advance is presumably independent of the similar condition in vertebrate erythrocytes. Kindred (1924) describes the presence of hemocytes or nucleated red cells in the perivisceral fluid of the Holothuroidea. They seem to be present in this group in general, with the exception of those forms which have tests, such as Psolus (The'el, 1920). Kindred did find them in Cucurnaria but not in Stichopus. We also have studied the perivisceral fluid of Stichopus, and have not found "red cells" there. In Cucumaria they are flattened biconvex discs with small, eccentric nuclei. They are orange-yellow in color. Van der Heyde (1922) made a study of such cells in Thy one briareus and by spectroscopic means was able to identify oxyhemoglobin and hemoglobin. He also obtained hemin-like crystals. It seems definite, then, that some of the echinoderms have red blood corpuscles carrying a respiratory pigment. It seems possible that in some forms such cells are confined to the water-vascular system, but they apparently are present in the general perivisceral (coelomic) fluid and in the hemal system in a number of species. Kindred (1924) believes that these cells have appeared with the development of the highly developed muscular system in the sea-cucumbers. This system requires more oxygen than the coelomic fluid alone could supply. Probably, there are two main categories of cells in all echinoderms: (1) ameboid phagocytic leucocytes, which have no noncytoplasmic inclusions except obviously phagocytized foreign material; and (2) spherule cells (trephocytes of Liebman, 1950) which contain conspicuous, often colored, inclusions, apparently characteristic of the cells. Both of these types are actively ameboid. Our observations have been made on Holothuroidea, Echinoidea, and Ophiuroidea. Holothuroidea. The types of cells vary in number and even to some extent in kind in different species, but some are common to all. The red blood cells, or hemocytes, when present, are relatively abundant. Thus in Paracaudina, there are 150,000/mm 3 of coelomic fluid (Okazaki and Koizumi, 1926). Among the ameboid cells those with colorless spherules are widespread and numerous. They usually are found in abundance in the body wall (Figs. 5, 6 and 9). We have watched such cells in active ameboid progression in fresh pieces of body wall and of intestine of Stichopus badionotus. They can be seen to enter and leave sinusoids in much the same manner as do vertebrate leucocytes. Cells with colored spherules, usually yellow, also are found and have been considered by some to be excretory in nature. Various types of leucocytes have been described, including, besides the more ordinary phagocytes, homogeneous and vesicular ones. "Crystal cells," cells almost filled by one large crystal, have been noted by Theel(1920), Kawamoto (1927) and Ohuye (1934 and 1936). Less common are spindleshaped cells, long fusiform elements. Kindred (1924) describes minute vibratile cells with flagella in the coelomic fluid of Stichopus californicus, but did not find these in two species of Cucumaria studied. We have failed to find such cells in S. badionolus. Conspicuous in the coelomic fluid of the holothuroideans are the petaloid leucocytes. In these the ectoplasm extends outward as broad, transparent lamellae which apparently aid the cell to float in the coelomic fluid while the endoplasm, relatively small in amount, contains the nucleus. These cells are phagocytic and with their

9 CELLS OF FLUIDS IN TUNICATES AND ECHINODERMS 293 peculiar arrangements can be "three-dimensionally" so in the body fluid. The coelom often contains small clumps known as broiun bodies. They may consist exclusively of cells with colored spherules (Becher, 1907), or they may also contain parasites, especially gregarines, and other types of ejecta. They seem to be excretory or waste material. As to the origin of the coelomocytes, Prosser and Judson (1952) believe that loci of formation of such cells are seen among mesenchyme cells in the walls of the haemal vessels. Cuenot (1891) thought that the polian vesicles were an important source of such cells. According to most descriptions, the channels of the haemal system in holothuroids lack a lining and perhaps are better referred to as sinuses or lacunae. Externally they are covered by coelomic epithelium, as they are surrounded by the perihemal sinuses or extensions of the coelom. In some places, as in the intestinal wall, the blood channels are simply spaces in the inner connective tissue layer. However, Prosser and Judson (1952) speak of a "thin, unicellular layer of endothelium" in Stichopus californicus. The relationship of the three systems in which body fluids are found in the echinoderms is of interest. Concerning the holothurians, Hyman, summarizing the findings of other authors, says (1955, p. 169): "It appears, however, that the blood spaces of the haemal ring are often continuous with the lumen of the water ring and even with the peripharyngeal sinus. Such communications allow an exchange of coelomocytes and fluid between the coelom and haemal and water-vascular systems." The water-vascular system contains noncellular formed elements of brownish color, often visible to the naked eye. A very small quantity of dissolved protein is found in the polian vesicles. The fluid contained in the blood channels is colorless in many holothurians but in some species is brown, in others yellow, and in still others rose-red. According to Durham (1892), the red color is due to the presence of the hemocytes. There apparently is no constant or wellregulated "circulation" of blood, nor a good reason to speak of "arteries and veins." Contractions of muscle fibers in the walls of the larger vessels, movements of the intestine, and contractions of the body wall move the blood in an irregular and somewhat pendulum-like fashion. The rather well marked intestinal vessels apparently are developed for collection of the absorbed food material; and they pass it on to the extensive lacunar system of the connective tissue. Durham (1892) felt that the branches of the blood or vascular system served like "railway" lines for the ameboid spherular cells to use in carrying nourishment to the various parts of the body. Cuenot (1891) believed that the phagocytic leucocytes became "mulberry-shaped" or "reserve cells" by taking up of food materials and then distributed these to the tissues. This was essentially the later view of Kindred (1926). The morphological elements, cellular and non-cellular, of the coelomic cavity appear to be practically the same as those of the water-vascular system. It is surprising that the site and manner of origin of such distinctive cells as the red hemocytes of holothurians have not yet been worked out. Ophiuroidea. Foettinger (1880a, b) described the discovery of hemoglobin in a small brittle star Ophiactis virens, from the Gulf of Naples. He identified oxyhemoglobin, by spectroscopic methods, in the "red blood cells" of the ambulacral system of this organism. These were yellowish-red cells which, en masse in the vessels, give a red color. They lack nuclei! While for the most part spheroidal, many of them are in the form of biconcave discs and present a center brighter than the periphery. They often appear slightly deformed when pressed upon by others. Size was found to

10 294 WARREN ANDREW be quite variable, from 1.5 ^ to 10.5 p. or on up to "giant blood corpuscles" of 20 p. diameter, although the latter are not common. The identity of the spectroscopic picture of the pigment in these cells with that of hemoglobin is not sufficient to prove its identity with that pigment, as some other substances, including turacin and helicorubin seem to give the same spectroscopic results; and hemin crystals have not been obtained from these cells. However, it does seem to be a respiratory pigment similar to that of the vertebrates. According to Cuenot (1891, 1897), the red corpuscles are found, along with numerous amoebocytes, in the ambulacral system but not in the coelomic cavity, which contains only amoebocytes. This must mean that the ambulacral system carries on a blood-respiratory function in this animal and that it is shut off both from the external sea-water and also from the coelomic cavity. The coelomocytes include leucocytes with slender pseudopods that tend to anastomose and form networks and spherule cells. The latter have been pictured in the tissues in the epidermis and just beneath it in Ophiactis (Cue"not, 1891). Our own observations confirm the tendency of the pseudopods to anastomose, and to give the cell a plate-like appearance. We examined coelomic fluid of Ophionereis reticulata. The spherule cells are few in number and those seen by us were colorless. Echinoidea. The coelomic fluid of the sea-urchins is an abundant, slightly turbid fluid with a specific gravity approximately the same as that of sea-water (1.026), with a small amount of dissolved protein, and with numerous cells. The cells are of several different types. The regular leucocytes show long, threadlike, branching pseudopodia. They are numerous, as are also the spherule-containing cells. These latter have the cytoplasm almost completely filled with large spherules, which may be colorless, green, or red. These cells move in a manner such as to give them the appearance of a "rat-tail maggot" (Liebman, 1950) in which the cell appears as a bulky ovoid structure, packed with spherules, with a short "tail" posteriorly, generally consisting of clear cytoplasm. Liebman made counts on the types of cells in the coelomic fluid of a common urchin, Arbacia punctulaia. He found 50.4% of the cells to be of the spherule-containing type (or perhaps, better, "inclusion" type), 49.6% to be phagocytes, or phagocytic leucocytes. Liebman (1950) found the red cells (not respiratory in nature) to comprise 48% of the total of those cells containing included material; the colorless ones, 41.8%; and the green ones, 10.2%. The cells which contain the red material, or "mahogany-brown" substance, were described by Geddes ( ) as showing a rather rapid and different type of motion as compared to those carrying material of other color. The substance itself also was described as amorphous, although later authors (Liebman, 1950) have pictured the red substance as spherular. In the coelomic fluid of Lytechinus variegatus atlaniicus, we have found the red material to appear amorphous and to seem to flow very readily within the cells, which progress by a rapid ameboid motion in cover-glass preparations. It was the opinion of Geddes ( ) that the yellowgreen spherules undergo a transformation into the amorphous reddish substance, and Liebman thinks this to be probable. The amorphous reddish substance has been shown to contain considerable iron. Cue'not (1891) found the amebocytes of the urchins, like those of sea stars, to have a slightly acid reaction (as opposed to the slightly alkaline reaction of the fluid itself), which would be in accord with their phagocytic and, according to him, excretory functions in both groups. The red-brown substance has been named

11 CELLS OK FLUIDS IN TUNICATES AND ECHINODKRMS 295 echinochrome, and many chemical studies have been made upon it. Cuenot believed it to be a fat in combination with a pigment. It is slightly soluble in alcohol, easily so in ether, chloroform, and benzine. It is weakly colored with osmic acid. Rosecolored, needle-shaped crystals have been obtained from it. The vibratile cells of earlier authors have been called "flagellated phagocytes" by Liebman. They resemble spermatozoa, but are much larger, and the "heads" are spheroidal, with a large nucleus and scanty cytoplasm. The tail is highly active and these cells must keep the coelomic fluid in a state of constant motion. Such cells have been found in all sea-urchins. The capacious body cavity of these organisms might not be served adequately by the ciliated coelomic epithelium which lines it, and may require additional means of circulation. We have observed that the reddish-brown cells show discrete spherules in Mellitn quinquiesperforala, while in sea-urchins the contents are amorphous. THE PROBLEM OF THE "TREPHOCYTKS" IN ECHINODERMS AND TUNICATES The echinoderms present excellent examples of the cells which Liebman (1945b, 1946, 1947, and 1950) has described as "trephocytes" or nourishing cells; for the cells with inclusions, often called the spherule cells, would pass muster as trephocytes. Indeed, Liebman has pointed out that in the ovaries of Arbacia punclulala large numbers of these cells can be seen in process of disintegration, and they presumably scatter nutritive materials about in the tissue. Tn fact, the developing eggs themselves are described as actively ingesting large droplets, "some still showing unmistakable marks of nuclei." He believes that a considerable proportion of the substance of the egg is thus taken up from the trephocytes, and that the echinochrome of the eggs may well have its origin from the red or "mahogany-brown" trephocytes. He points out also that uptake and digestion of whole trephocytes by the growing ovarian eggs are apparently common phenomena in the invertebrates, as in Tubidaria among the coelenterates, and in the tunicates. Trephocytes are also seen frequently within phagocytic cells in Arbacia. It seems probable that they distribute the nourishing substances widely through the body. Liebman believes that the trephocytes have not been shown to have any genetic relationship to the different kinds of phagocytes and that this tends to prove the distinctive nature of each type. DISCUSSION AND CONCLUSIONS Our studies on the blood cells of tunicates have impressed us with the similarities of some of the types in these animals to the spherule cells (trephocytes) of the echinoderms. We refer particularly to the green cells and to their colorless counterparts. These colorless counterparts, however, we do not believe should be confined to the colorless cells of green cell type but should be expanded to include the "colorless morula" cells which contain inclusion bodies giving the cells a berry-like appearance. It seems to us significant that of the 26 species of tunicates studied by George (1939), he lists only one, Ecleinascidia turbinata as having both the colorless cells of green type and the colorless morula type. It appears to us that there is a strong probability that these very similar cells, in different species, might be classed in some cases as colorless cells of green type, in others as colorless morula cells. As far as the reaction with osmic acid is concerned, while the colorless morula cells are described as negative, the reaction of the green cells is said to be negative in some species, positive in others. This variation, George believed, might be due to absence of vanadium in some, or to its presence in the form of a high oxide which would not reduce osmic acid. In any event, it does not appear as a constant criterion for distinguishing between the green cells (of

12 296 WARREN ANDREW green or "colorless" type) and the colorless morula cells. Our own observations with oil immersion and phase microscopy have impressed us with the morphological similarities between colorless morula and green cells. In these cells the included bodies appear in the resting cell as angular or somewhat wedge-shaped structures, very sharply set off from the surrounding cytoplasm. We have not observed the colorless morula cells to be motile in any of our cover-slip preparations. On the other hand, the green cells, while seen to be actively motile in some locations in the tissues, as in the transparent test of Ecteinascidia turbinata, are only very sluggishly so in coverslip preparations. In a comparison of the blood cells of tunicates with those of the coelomic fluid and blood of other invertebrates, and in particular of the echinoderm's, it seems to us probable that the green cells, both green and "colorless," and the colorless morula cells, are to be classed as trephocytes like the spherule cells of echinoderms. It should be recalled here that the latter cells include, in various species, cells with spherules of different colors and cells with colorless spherules. We feel that the concept of earlier authors that wandering cells simply carry ingested (phagocytized) food to the tissues is probably an over-simplification. We do think it likely that such cells develop within their cytoplasm stored food materials, perhaps of various kinds, the materials for which they have received by phagocytosis but which become in them a finished product suitable for distribution to other cells. If these views have validity, some of the large groups of cells of blood and coelomic fluid of tunicates and echinoderms are not too difficult to homologize. REFERENCES Andrew, W Phase microscope studies of living blood-cells of the tunicates under normal and experimental conditions, with a description of a new type of motile cell appendage. Quart. J. Microscop. Sci. 102: Arnold, J. W Observations of ameboid motion of living hemocytes in the wing veins of Blaberwi giganteus (L.). Canad. J. Zool. 37: Becher, S Rhabdomolgus ruber. Z. Wiss. Zool. 88: Botazzi, F (cited from Winterstein) Vergleichende Physiologie, Gustav Fischer, Jena. Cu^not, L Etudes sur le sang et les glands lymphatiques dans la se>ie animale (2* Partie: Invertebres). Arch. Zool. exp. gen. 9: Durham, H. E On wandering cells in echinoderms, more specially with regard to excretory functions. Quart. J. Microscop. Sci. (2), 33: Endean, R Studies on the blood and tests of some Australian ascidians. I. The blood of Pyura slolonifera Heller. Austr. J. Marine and Freshwater Research 6: Foettinger, A Sur l'existence de l'hemoglobine chez les echinodermes. Arch. Biol. 1: Fulton, J. F The blood of Ascidia atra l.esueur; with special reference to pigmentation and phagocytosis. Acta Zool. 1: Geddes, P Observations sur le fiuide perivisceral des oursins. Arch. Zool. exp. gen. 8: George, W. C The histology of the blood of Perophora viridis (ascidian). J. Morph. Physiol. 41: The histology of the blood of some Bermuda ascidians. J. Morph. Physiol. 49: A comparative study of the blood of the tunicates. Quart. J. Microscop. Sci. 81: Henze, M Untersuchungen iiber das Blut der Ascidien. I. Die Vanadiumverbingung der Blutkorpeichen. Hoppe-Seyl. Z. 72: Untersuchungen uber das Blut der Ascidien. II. Hoppe-Seyl. Z. 79: Untersuchungen uber das Blut der Ascidien. III. Mitteilung. Hoppe-Seyl. Z. 86: Hyman, L The Invertebrates. Echinodermata, Vol. 4. McGraw-Hill, New York. Kawamoto, N Anatomy of Caudina chilensis (J. Miiller) with special reference to the perivisceral cavity, the blood and the water vascular systems in their relation to the blood circulation. Sci. Repts. Tohoku Univ. ser. 4, 2:2S Kindred, J. E The cellular elements in the perivisceral fluid of echinoderms. Biol. Bull. 46: A study of the genetic relationships of the "amebocytes with spherules" in Arbacia. Biol. Bull. 26: Kollmann, M Recherches sur les leucocytes et le tissue lymphoi'des des invert^bres. Ann. Sci. Natur., Zool. ser. 9, 8: Liebman, E The function of leucocytes in the growth and regression of the egg of THturtts viridescem. Am. J. Anat. 77: On trephocytes and trephocytosis; a

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